Abstract
Polyploidization plays an important role in the genesis of cultivated wheat (hexaploid and tetraploid) from its diploid progenitors. Thus, evolution during polyploidization resulted in present-day hexaploid bread wheat. GS2 and Fd-GOGAT enzymes are core components involved in the assimilation of primary nitrogen (N) in plants. In the present study, we aimed to analyze these two important genes at their molecular level to find the extent of variation that occurred during evolution from the ancient diploid progenitors to the modern-day hexaploid bread wheat. Furthermore, we studied their gene expression pattern and assayed both the enzymes under N stress. We also investigated the degree of resilience among these species in terms of certain important morphophysiological and biochemical parameters under N stress. Comparison of the genomic sequences along with their promoter region revealed that both GS2 and Fd-GOGAT genes were located on chromosome 2 and were comprised of 13 and 33 exons respectively. A limited sequence divergence at cDNA and amino acid levels was observed among the genome species, but the divergence was significantly higher in the promoter region. Both these genes were present in three copies in the bread wheat, two copies in the durum wheat, and a single copy in their diploid progenitors. Differential gene expression among the five genome species under N stress suggested major differences in gene regulation due to the difference in cis-elements. Enzyme activity did not correlate with the gene expression level probably due to post-transcriptional and post-translational modification of the enzymes. There was neither correlation between the GS2 and Fd-GOGAT activity in any species. All growth parameters, except root length, decreased or remain unchanged with different degrees of plasticity in the genotypes under N stress and correlated with reduced Fd-GOGAT activity, which supply the primary assimilate glutamate. GS2/Fd-GOGAT enzyme activity along with total N accumulation, protein, chlorophyll, and carotenoid content in shoot were found responsive to the N stress through combined PCA analysis. Our study confirmed the conserved nature of GS2 and Fd-GOGAT enzymes at the CDS and protein level; however, their expression and subsequent effects were different in cultivated wheat and their progenitors.
Similar content being viewed by others
Change history
13 November 2021
A Correction to this paper has been published: https://doi.org/10.1007/s11105-021-01322-6
References
Andrews M, Lea PJ, Raven JA, Lindsey K (2004) Can genetic manipulation of plant nitrogen assimilation enzymes result in increased crop yield and greater N-use efficiency? An assessment. Ann Appl Biol 145:25–40. https://doi.org/10.1111/j.1744-7348.2004.tb00356.x
Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. https://doi.org/10.1093/bioinformatics/bti770
Batten GD (2017) The uptake and utilization of phosphorus and nitrogen by diploid, tetraploid and hexaploid wheats (Triticum spp.). Ann Bot 58:49–59. https://doi.org/10.1093/oxfordjournals.aob.a087187
Benkert P, Kunzli M, Schwede T (2009) QMEAN server for protein model quality estimation. Nucleic Acids Res 37:510–514. https://doi.org/10.1093/nar/gkp322
Brooks SA, Huang L, Herbel MN, Gill BS, Brown-Guedira G, Fellers JP (2006) Structural variation and evolution of a defense-gene cluster in natural populations of Aegilops tauschii. Theor Appl Genet 112:618–626. https://doi.org/10.1007/s00122-005-0160-7
Consortium (IWGSC), TIWGS, Investigators, I R principal, Appels R, Eversole K, Feuillet C, Keller B, Uauy C et al (2018) Shifting the limits in wheat research and breeding using a fully annotated reference genome. Science 361(6403): eaar7191. https://doi.org/10.1126/science.aar7191
Cormier F, Foulkes J, Hirel B, Moenne-Loccoz Y, Gouache D, Allard V, Le Gouis J (2016) Breeding for increased nitrogen use efficiency : a review for wheat. Plant Breed 135:255–278
Coucepcion A, Antonio JM, Purificacion P, Martin EC, Roger MW, Brian GF (1993) Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme. Planta 189:475–483
Cren M, Hirel B (1999) Glutamine synthetase in higher plants: regulation of gene and protein expression from the organ to the cell. Plant Cell Physiol 40:1187–1193. https://doi.org/10.1093/oxfordjournals.pcp.a029506
Dechorgnat J, Nguyen CT, Armengaud P, Jossier M, Diatloff E, Filleur S, Daniel-Vedele F (2011) From the soil to the seeds: the long journey of nitrate in plants. J Exp Bot 62(4):1349–1359. https://doi.org/10.1093/jxb/erq409
Dehzangi A, Paliwal K, Lyons J, Sharma A, Sattar A (2014) Proposing a highly accurate protein structural class predictor using segmentation-based features. BMC Genomics 15:1–13. https://doi.org/10.1186/1471-2164-15-S1-S2
DeLano WL (2002) The PyMOL molecular graphics system. San Carlos, DeLano Scientific California
Dvorak J, Akhunov ED (2005) Tempos of gene locus deletions and duplications and their relationship to recombination rate during diploid and polyploid evolution in the Aegilops-Triticum alliance. Genetics 171:323–332. https://doi.org/10.1534/genetics.105.041632
Eisenberg D, Gill HS, Pfluegl GMU, Rotstein SH (2005) Structure-function relationships of glutamine synthetasesre. Biochim Biophys Acta - Protein Struct Mol Enzymol 1477:122–145. https://doi.org/10.1016/S0167-4838(99)00270-8
Fathi G (2008) Effect of genotype variability on nitrate uptake and assimilation of wheat cultivars. J Agric Sci Technol 10:11–22
Foulkes MJ, Hawkesford MJ, Barraclough PB, Holdsworth MJ, Kerr S, Kightley S, Shewry PR (2009) Identifying traits to improve the nitrogen economy of wheat: recent advances and future prospects. F Crop Res 114:329–342. https://doi.org/10.1016/j.fcr.2009.09.005
Galloway JN, Aber JD, Erisman JW, Seitzinger SP, Howarth RW, Cowling EB, Cosby BJ (2006) The nitrogen cascade. Bioscience 53:341. https://doi.org/10.1641/0006-3568(2003)053[0341:tnc]2.0.co;2
Gayatri RM, Mahato AK, Sinha SK, Dalal M, Singh NK, Mandal PK (2018) Homeologue specific gene expression analysis of two vital carbon metabolizing enzymes—citrate synthase and NADP-isocitrate dehydrogenase—from wheat (Triticum aestivum L.) under nitrogen stress. Appl Biochem Biotechnol 188(3):569–584. https://doi.org/10.1007/s12010-018-2912-2
Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326
Gorny AG, Garczynski S (2008) Nitrogen and phosphorus efficiency in wild and cultivated species of wheat. J Plant Nutr 31:263–279. https://doi.org/10.1080/01904160701853878
Hernandez-Garcia CM, Finer JJ (2014) Identification and validation of promoters and cis-acting regulatory elements. Plant Sci 217-218:109–119. https://doi.org/10.1016/j.plantsci.2013.12.007
Hirel B, Bertin P, Quillere I, Bourdoncle W, Attagnant C, Dellay C, Gouy A, Cadiou S, Retailliau C, Falque M, Gallais A (2001) Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize. Plant Physiol 125:1258–1270
Hirel B, Le Gouis J, Ney B, Gallais A (2007) The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. J Exp Bot 58:2369–2387. https://doi.org/10.1093/jxb/erm097
Hiscox JT, Israelstam GF (1979) A method for the extraction of chlorophyll from leaf tissue without maceration. Can J Bot 57:1332–1334
Huang S, Sirikhachornkit A, Su X, Faris J, Gill B, Haselkorn R, Gornicki P (2002) Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase of the Triticum/Aegilops complex and the evolutionary history of polyploid wheat. Proc Natl Acad Sci 99:8133–8138. https://doi.org/10.1073/pnas.072223799
Idicula-Thomas S, Balaji PV (2005) Understanding the relationship between the primary structure of proteins and its propensity to be soluble on overexpression in Escherichia coli. Protein Sci 14:582–592
Kant S, Bi YM, Rothstein SJ (2011) Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency. J Exp Bot 62:1499–1509. https://doi.org/10.1093/jxb/erq297
Kumagai E, Araki T, Hamaoka N, Ueno O (2011) Ammonia emission from rice leaves in relation to photorespiration and genotypic differences in glutamine synthetase activity. Ann Bot 108:1381–1386. https://doi.org/10.1093/aob/mcr245
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132. https://doi.org/10.1016/0022-2836(82)90515-0
Li XP, Zhao XQ, He X, Zhao GY, Li B, Liu DC, Zhang AM, Zhang XY, Tong YP, Li ZS (2011) Haplotype analysis of the genes encoding glutamine synthetase plastic isoforms and their association with nitrogen-use- and yield-related traits in bread wheat. New Phytol 189:449–458. https://doi.org/10.1111/j.1469-8137.2010.03490.x
Ling HQ, Qiu J, Singh RP, Keller B (2004) Identification and genetic characterization of an Aegilops tauschii ortholog of the wheat leaf rust disease resistance gene Lr1. Theor Appl Genet 109:1133–1138. https://doi.org/10.1007/s00122-004-1734-5
Lowry OH, Niraj Rosebrough ARJR, Farr AL (1994) The folin by oliver. Anal Biochem 217:220–230. https://doi.org/10.1016/0304-3894(92)87011-4
Marquez AJ (2005) Lotus Japonicus handbook, 1st ed. Springer Dordrecht, The Netherlands 1–384
Marquez AJ, Avila C, Forde BG, Wallsgrove RM (1988) Ferredoxin-glutamate synthase from barley leaves: rapid purification and partial characterization. Plant Physiol Bioch 26:645–651
Martin F, Suzuki A, Hirel B (1982) A new high-performance liquid chromatography assay for glutamine synthetase and glutamate synthase in plant tissues. Anal Biochem 125:24–29. https://doi.org/10.1016/0003-2697(82)90378-5
Martin A, Lee J, Kichey T, Gerentes D, Zivy M, Tatout C, Dubois F, Balliau T, Valot B, Davanture M, Terce-Laforgue T, Quillere I, Coque M, Gallais A, Gonzalez-Moro MB, Bethencourt L, Habash DZ, Lea PJ, Charcosset A, Perez P, Murigneux A, Sakakibara H, Edwards KJ, Hirel B (2006) Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production. Plant Cell Online 18:3252–3274. https://doi.org/10.1105/tpc.106.042689
Menz J, Range T, Trini J, Ludewig U, Neuhauser B (2018) Molecular basis of differential nitrogen use efficiencies and nitrogen source preferences in contrasting Arabidopsis accessions. Sci Rep 8:1–11. https://doi.org/10.1038/s41598-018-21684-4
Migge A, Carrayol E, Caroline K, Bertrand H, Heinrich F, Thomas B (1997) The expression of the tobacco genes encoding plastidic glutamine synthetase or ferredoxin-dependent glutamate synthase does not depend on the rate of nitrate reduction, and is unaffected by suppression of photorespiration. J Exp Botany 48:1175–1184
Migge A, Carrayol E, Hirel B, Becker TW (2000) Leaf-specific overexpression of plastidic glutamine synthetase stimulates the growth of transgenic tobacco seedlings. Planta 210:252–260. https://doi.org/10.1007/PL00008132
Nigro D, Gu YQ, Huo N, Marcotuli I, Blanco A, Gadaleta A, Anderson OD (2013) Structural analysis of the wheat genes encoding NADH-dependent glutamine-2-oxoglutarate amidotransferases and correlation with grain protein content. PLoS One 8:1–11. https://doi.org/10.1371/journal.pone.0073751
Nigro D, Blanco A, Anderson OD, Gadaleta A (2014) Characterization of ferredoxin-dependent glutamine-oxoglutarate amidotransferase (Fd-GOGAT) genes and their relationship with grain protein content QTL in wheat. PLoS One 9(8):e103869. https://doi.org/10.1371/journal.pone.0103869
Obara M, Sato T, Sasaki S, Kashiba K, Nagano A, Nakamura I, Ebitani T, Yano M, Yamaya T (2004) Identification and characterization of a QTL on chromosome 2 for cytosolic glutamine synthetase content and panicle number in rice. Theor Appl Genet 110:1–11. https://doi.org/10.1007/s00122-004-18280
Pandey B, Saini M, Sharma P (2016) Molecular phylogenetic and sequence variation analysis of dimeric α-amylase inhibitor genes in wheat and its wild relative species. Plant Gene 6:48–58. https://doi.org/10.1016/j.plgene.2016.03.004
Pour-Aboughadareh A, Mahmoudi M, Moghaddam M, Ahmadi J, Mehrabi AA, Alavikia SS (2017) Agro-morphological and molecular variability in Triticum boeoticum accessions from Zagros Mountains, Iran. Genet Resour Crop Evol 64:545–556. https://doi.org/10.1007/s10722-016-0381-4
Remmert M, Biegert A, Hauser A, Soding J (2012) HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods 9:173–175. https://doi.org/10.1038/nmeth.1818
Rigano C, Di Martino RV, Vona V, Carfagna S, Carillo P, Esposito S (1996) NH4+ assimilation by roots of young barley plants, changes in pool of free glutamine and asparagine and respiratory oxygen consumption. Plant Phys and Bioch 34:683–690
Rowe WB, Ronzio RA, Wellner YP, Meister A (1970) Glutamine synthetase (sheep brain). in: Methods in enzymology, Part A 900–910
Sinha SK, Rani M, Bansal N, Gayatri VK, Mandal PK (2015) Nitrate starvation induced changes in root system architecture, carbon:nitrogen metabolism, and miRNA expression in nitrogen responsive wheat genotypes. App Bioch and Biotech 6:1299–1312
Sinha SK, Amitha Mithra SV, Chaudhary S, Tyagi P, Venkadesan S, Rani M, Mandal PK (2018) Transcriptome analysis of two rice varieties contrasting for nitrogen use efficiency under chronic N starvation reveals differences in chloroplast and starch metabolism-related genes. Genes (Basel) 9:1–22. https://doi.org/10.3390/genes9040206
Stewart CN, Via LE (1993) A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques 14:748–750
Stitt M, Scheible WR (1999) Nitrate acts as a signal to control gene expression, metabolism and biomass allocation, in: Kruger N, Hill, SA, Ratcliffe RG, (Eds.), regulation of metabolism. Dordrecht: Kluwer Academic Publishers 275–306
Swarbreck SM, Defoin-Platel M, Hindle M, Saqi M, Habash DZ (2011) New perspectives on glutamine synthetase in grasses. J Exp Bot 62:1511–1522. https://doi.org/10.1093/jxb/erq356
Tobin AK, Yamaya T (2001) Cellular compartmentation of ammonium assimilation in rice and barley. J Exp Bot 52:591–604. https://doi.org/10.1093/jxb/52.356.591
Unno H, Uchida T, Sugawara H, Kurisu G, Sugiyama T, Yamaya T, Sakakibara H, Hase T, Kusunoki M (2006) Atomic structure of plant glutamine synthetase. J Biol Chem 281:29287–29296. https://doi.org/10.1074/jbc.m601497200
Van den Heuvel RH, Ferrari D, Bossi RT, Ravasio S, Curti B, Vanoni MA, Florencio FJ, Mattevi A (2002) Structural studies on the synchronization of catalytic centers in glutamate synthase. J Biol Chem 277:24579–24583
Wray GA, Hahn MW, Abouheif E, BalhoV JP, Pizer M, Rockman MV, Romano LA (2003) The evolution of transcriptional regulation in eukaryotes. Mol Biol Evol 20:1377–1419
Yang X, Nian J, Xie Q, Feng J, Zhang F, Jing H, Zhang J, Dong G, Liang Y, Peng J, Wang G, Qian Q, Zuo J (2016) Rice ferredoxin-dependent glutamate synthase regulates nitrogen–carbon metabolomes and is genetically differentiated between japonica and indica subspecies. Mol Plant 9:1520–1534. https://doi.org/10.1016/j.molp.2016.09.004
Yang C, Yang Z, Zhao L, Sun F, Liu B (2018) A newly formed hexaploid wheat exhibits immediate higher tolerance to nitrogen-deficiency than its parental lines. BMC Plant Biol 18:1–12. https://doi.org/10.1186/s12870-018-1334-1
Zhang H, Zhu B, Qi B, Gou X, Dong Y, Xu C, Zhang B, Huang W, Liu C, Wang X, Yang C, Zhou H, Kashkush K, Feldman M, Wendel JF, Liu B (2014) Evolution of the BBAA component of bread wheat during its history at the allohexaploid level. Plant Cell 26:2761–2776. https://doi.org/10.1105/tpc.114.128439
Zhang W, Fan X, Gao Y, Liu L, Sun L, Su Q, Han J, Zhang N, Cui F, Ji J, Tong Y, Li J (2017) Chromatin modification contributes to the expression divergence of three TaGS2 homoeologs in hexaploid wheat. Sci Rep 7:44677. https://doi.org/10.1038/srep44677
Acknowledgments
We are thankful to Dr. K. Venkatesh, Dr. B. S. Tyagi (IIWBR), and Dr. Anju Mahendru Singh (ICAR-IARI) for providing seed material and Director, NIPB, New Delhi for providing all the facilities. The authors also want to thank Arivaradarajan Preeti for helping in editing the manuscript.
Funding
This research was supported by the Newton Bhaba Fund project “Indo-UK Centre for the improvement of nitrogen use efficiency in wheat” (INEW) co-funded by the Department of Biotechnology, Govt. of India (BT/IN/UK-VNC/43/KV/2015–16).
Author information
Authors and Affiliations
Contributions
G designed and performed the experiments. KJ performed southern blot analysis. SKS and PR revised the manuscript. PKM designed the experiments, revised the manuscript, and supervised the entire work as Project Investigator.
Corresponding author
Ethics declarations
Conflict of Interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key message
Comprehensive analysis of GS2 and Fd-GOGAT genes revealed difference in copy number and gene expression profile among the polyploid cultivated species of wheat and their diploid progenitors, in spite of near conserved protein structure across the genome species.
Rights and permissions
About this article
Cite this article
Gayatri, Jayaraman, K., Sinha, S.K. et al. Comparative Analysis of GS2 and Fd-GOGAT Genes in Cultivated Wheat and Their Progenitors Under N Stress. Plant Mol Biol Rep 39, 520–545 (2021). https://doi.org/10.1007/s11105-020-01267-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11105-020-01267-2